This is a renewal application for support for year 27 of Cell Transformation by Bovine Papillomavirus (R01 CA37157). This grant supported the discovery of the unique mechanism of action of the bovine papillomavirus E5 oncoprotein, a 44-amino acid very hydrophobic protein that is essentially an isolated transmembrane domain. We showed that the E5 protein binds to the transmembrane domain of the PDGF ? receptor, resulting in ligand-independent receptor activation. Detailed analysis of this interaction is providing new insights into transmembrane domain recognition and allowed us to develop a novel approach to manipulate cell behavior with artificial, small transmembrane proteins modeled on the viral protein. In an exciting advance in this funding period, we reprogrammed the E5 protein so that it no longer recognizes the PDGF receptor, but instead specifically activates the human erythropoietin receptor, driving erythroid differentiation of human hematopoietic progenitor cells. We propose here to extend our in-depth analysis of the interaction of the E5 protein and the PDGF ? receptor, with a particular attention to obtaining novel insights into the molecular basis of specific protein recognition within cell membranes, a process that underlies numerous biological processes, including many cancers. We will also develop small transmembrane proteins as potent biological agents against cancer and related diseases. We will isolate E5 mutants that recognize PDGF ? receptor mutants, to determine the rules responsible for highly-specific interactions between transmembrane helices. We will use related approaches to identify and characterize E5 mutants that inhibit PDGF ? receptor signaling. We will construct and analyze peptides to characterize the interaction between the E5 protein and the PDGF ? receptor transmembrane domain at the biochemical level. Finally, we will isolate and characterize small transmembrane proteins that modulate the HIV co-receptor, CCR5, a multipass transmembrane protein. These experiments should continue to provide important new insights into an unusual viral oncogene product and protein recognition within cell membranes, as well as providing new approaches to modulate important biological processes.

Public Health Relevance

This grant supported the discovery of the unique mechanism of action of a cancer-causing protein from an animal papillomavirus. Continuing analysis of this mechanism will provide new molecular insight into a class of protein interactions that control cancer and other important biological processes. In addition, on the basis of these studies, we will continue to develop a novel protein engineering method to control biological processes, including infection by human immunodeficiency virus, an important co-factor in many cancers.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA037157-30
Application #
8445311
Study Section
Virology - A Study Section (VIRA)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1984-04-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
30
Fiscal Year
2013
Total Cost
$462,671
Indirect Cost
$182,637
Name
Yale University
Department
Genetics
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Cohen, Emily B; Jun, Susan J; Bears, Zachary et al. (2014) Mapping the homodimer interface of an optimized, artificial, transmembrane protein activator of the human erythropoietin receptor. PLoS One 9:e95593
Dimaio, Daniel (2014) Is virology dead? MBio 5:e01003-14
DiMaio, Daniel (2014) Viral miniproteins. Annu Rev Microbiol 68:21-43
Chacon, Kelly M; Petti, Lisa M; Scheideman, Elizabeth H et al. (2014) De novo selection of oncogenes. Proc Natl Acad Sci U S A 111:E6-E14
Edwards, Anne P B; Xie, Yanhua; Bowers, Lara et al. (2013) Compensatory mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor * receptor reveal a complex direct transmembrane interaction. J Virol 87:10936-45
DiMaio, Daniel; Petti, Lisa M (2013) The E5 proteins. Virology 445:99-114
Petti, Lisa M; Talbert-Slagle, Kristina; Hochstrasser, Megan L et al. (2013) A single amino acid substitution converts a transmembrane protein activator of the platelet-derived growth factor * receptor into an inhibitor. J Biol Chem 288:27273-86
Marlatt, Sara A; Kong, Yong; Cammett, Tobin J et al. (2011) Construction and maintenance of randomized retroviral expression libraries for transmembrane protein engineering. Protein Eng Des Sel 24:311-20
Cammett, Tobin J; Jun, Susan J; Cohen, Emily B et al. (2010) Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor. Proc Natl Acad Sci U S A 107:3447-52
Talbert-Slagle, Kristina; DiMaio, Daniel (2009) The bovine papillomavirus E5 protein and the PDGF beta receptor: it takes two to tango. Virology 384:345-51

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